Project Summary Mycobacterium tuberculosis (Mtb) infects one-third of the world's population and claims 1.5 million lives each year. Although antibiotics can rapidly kill Mtb in vitro, the current standard of care for tuberculosis (TB) is six months of treatment. Understanding this discrepancy is a vital step towards shortening TB therapy. Because antibiotics generally target growth-related processes, it has been postulated since the 1950s that a small population of Mtb with downshifted replication is responsible for antibiotic tolerance and, therefore, prolonged treatment. More recent evidence suggests that mycobacterial growth rates are heterogeneous and dynamic within and between the lesions present at different stages of infection. The nature of Mtb replication ? homogeneous or heterogeneous, static or dynamic ? also has practical implications for monitoring since the response of slow or nongrowing Mtb to treatment may indicate efficacy better than standard, bulk measurements of bacterial burden. Although the importance of Mtb growth is widely accepted, it has not been possible to directly test most hypotheses related to in vivo and ex vivo antibiotic susceptibility. Current methods for evaluating bacterial replication overlook subpopulation variability or are ill suited to the complexities of host tissues or patient specimens. To address this critical technical hurdle, we have developed several strategies to tag the Mtb cell envelope via metabolic labeling and bioorthogonal chemical detection. Because growth of a cell requires expansion of its surface, new cell envelope provides a facile marker for single-cell replication. Pulse chase experiments with probes that are covalently affixed to the bacterial cell surface provide a tree ring- like record of growth over time. Individual rings can be functionalized with different labels, enabling fluorescent detection or purification of Mtb cells that differ in growth history. Using these strategies, we seek to build a comprehensive, predictive model for Mtb growth in response to host and antibiotic pressure. We hypothesize that 1) slow or nonreplication of Mtb subpopulations is the primary determinant of drug tolerance during infection and 2) tracking these subpopulations will speed predictions of antibiotic efficacy. The results of these experiments could fundamentally shape our view of Mtb infection and drug susceptibility and allow us to devise better therapeutic strategies for a pathogen of global public health import. !